Nutritional composition and phytochemical screening in different parts of Hibiscus syriacus L.

Abstract As the national flower of Korea, the Hibiscus syriacus L. (Rose of Sharon) is symbolic in its abundance and is a prominent feature of Korean culture. H. syriacus has played an important role in Korea, not only as an ornamental plant but also as an essential ingredient in folk remedies through its various parts. This study aimed to characterize the nutritional and biochemical composition of each plant unit of H. syriacus “Wonhwa.” The units are namely: the petals, leaves, roots, and sprouts from its seeds. According to the results each unit produced, the sprouts had the highest content of amino acids and fatty acids which adhere to the requirements of nutritionally excellent food ingredients. The petals produced high quantities of glucose, sucrose, and fumaric acid, with the highest antioxidant activity among the four units. The main bioactive compounds detected in H. syriacus extracts in the four units were o‐coumaric acid, p‐coumaric acid, schaftoside, isoschaftoside, apigenin‐6‐C‐glucoside‐7‐o‐glucoside, and kaempferol‐3‐O‐galactoside‐7‐O‐rhamnoside. Overall, the highest number of bioactive compounds, 2 phenolic acids and 22 flavonoids, were identified in the petals. These results suggest the possibility of excellent pharmacological activity in the petals.

Previous phytochemical investigations on this plant have resulted in the isolation of several naphthalenes, lignans, and flavonoids from root barks (Lee et al., 1999;Ryoo et al., 2010;Yoo et al., 1998). Recently, the antiproliferative activity of the root bark of this plant against cancer cells has been reported, and several triterpenoids were isolated as active constituents (Yeon et al., 2019).
At present, there are many published studies on the pharmacological activity of the root and bark. Yet, there is less on the compounds and pharmacological effects of the other parts of the plant.
Recently, two studies reported that the petal extracts of H. syriacus inhibit melanin, which is the cause of spots and freckles in the skin (Karunarathne et al., 2019). The young leaves of H. syriacus have also shown effects of in vitro anti-inflammatory properties (Eo et al., 2020).
Although studies thus far have proven the pharmacological ef-

| Amino acid determination
One gram of freeze-dried sample was weighed, and 30 ml of 80% methanol aqueous solution was used to extract these samples with sonication for 1 h. The extract was then filtered with a 0.2 μm membrane filter. The chromatic method were by the Agilent method (John et al., 2000). For derivatization of amino acids, the following reagents were used: 0.1 M borate buffer in water with 3-mercaptopropionic acid (3-MPA)/o-phthalaldehyde (OPA) reagent and 9-fluorenylmethyl chloroformate (FMOC-Cl) reagent. Derivatization was performed using an automatic injector with a successive sampling of 5.0 μl of borate buffer and 1.0 μl of sample and then mixed. Subsequently, 1.0 μl of 3-MPA/OPA reagent was added. After mixing, 1.0 μl of FMOC-Cl was added and mixed, followed by 32 μl of water. Separations and analysis were performed with Dionex Ultimate 3000 (Thermo Dionex) equipped with an Agilent 1260 Infinity Fluorescence (FL) detector (Agilent). The chromatographic column was an Inno C18 column (4.6 mm × 150 mm, 5 µm, Youngjin Biochrom), at 40°C, with detection at λ = 340 nm.
The amino acid determination was performed with Mobile Phase A, consisting of 40 mM NaH2PO4, adjusted to pH 7.0, and Mobile phase, which was acetonitrile/methanol/water (45/45/10 v/v/v %), and a flow rate of 1.5 ml/min. The initial condition was 95% A of A for 3.0 min. 5% of B, followed by a linear gradient to 55%, 90% for 1 min, and 90% of B isocratically for 6 min. And then, washing and equilibration at 5% of B were performed.
To calculate the calibration curves, four different points were obtained using the amino acid standard mixture. The results are expressed in mg/100 g of dry weight.

| Fatty acid determination
Samples of 100 mg with pentadecanoic acid (15:0) as the internal standard were placed in 4ml glass vials with Teflon caps.
Then, two phases formed. The upper phase containing FAMES was collected by GC (Agilent 7890A, Agilent). An Agilent DB-23 (120 m × 0.25 mm × 0.25 µm) column was used with an injector temperature and FID detector temperature of 250 and 280°C, respectively, and a carrier gas of He. The split ratio was set to 10:1.
The temperature program was as follows: 50°C for 1.0 min, increase at 25°C/min to 130°C, at 8°C/min to 170°C, at 1.5°C/min to 215°C, and at 5°C/min to 250°C for 5 min.

| Free sugar determination
One gram of freeze-dried sample and 30ml of 80% methanol aqueous solution were used to extract these samples with sonication for 1 h. The extract was filtered in a 0.2 μm membrane filter. The separations and analyses were performed with an HPLC system (Dionex ultimate 3000, Thermo Dionex), consisting of a Shodex Refractive Index (RI)-101 detector (Shodex), a column heater set at 70°C, and a sugar-pak column (300 × 6.5mm, Waters); the isocratic mobile phase was deionized-distilled H2O delivered at 0.5 ml/min. The calibration curve with four different points was used to obtain the free sugar standard. The results are in mg/100 g of dry weight.

| Organic acid determination
One gram of freeze-dried sample was weighed. And a 30 ml of 80% methanol aqueous solution was used to extract these samples with sonication for 1 h. The extract was filtered with a 0.2 μm membrane filter.
The HPLC system (Ultimate 3000, Thermo Dionex) with a pump system and an RI detector (ERC, RefractoMAX520) monitoring at 210 nm were used for organic acids analysis. Organic acids were simultaneously analyzed on an Aminex 87H column (300 × 10 mm, Bio-Rad) at 40°C with a flow rate of 0.5ml/min and eluent of 0.01N H2SO4. Four different points were obtained using organic acid standards to calculate the calibration curves. The results are in mg/100 g of dry weight.

| Total polyphenol content (TPC)
The extract was filtered with a 0.2 μm membrane filter. And 0.5 ml of distilled water was added to 100 μl of the extracted sample solution.
Then, 100 μl of Folin & Ciocalteu phenol reagent was added and mixed.
Subsequently, 1.0 ml of 7% Na2CO3 solution was added to the reaction solution. This mixed solution was allowed to stand at room temperature for 30 min. The absorbance was measured at 760 nm using a UV-VIS spectrophotometer (Biomate5 spectrophotometer, Thermo Scientific). The total polyphenol content of the extracts was expressed as mg gallic acid equivalents (GAE) per gram of sample in dry weight.

| ABTS radical cation scavenging activity
Ten grams of freeze-dried sample was weighed. And 200 ml of 70% ethanol aqueous solution was used to extract these samples with sonication for 48 h. The ABTS assay was performed according to the method described by Li et al. (2009) with slight modification. The 2,2-azo-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich) radical cation (ABTS+) was produced by mixing 7 mM ABTS solution with 2.45 mM potassium persulfate aqueous solution in the dark at room temperature for 16 to 24 h. The ABTS+ solution was diluted with phosphate buffer to the absorbance of 0.70 ± 0.02 at 732 nm (Perkin Elmer). Briefly, 90 μl ABTS solution and 10 μl of the sample were mixed and incubated at room temperature for 10 min. After 10 min, the absorbance was measured at 732 nm. For the blank, 10 μl DMSO was used instead of the sample. All tests were carried out in triplicate. The ABTS radical cation scavenging activity was calculated as follows: ABTS radical cation scavenging activity (%) = [1 − (Ai − Aj) / A0] × 100, where A0 is the absorbance of the blank sample, Ai is the absorbance in the presence of the sample at different concentrations, and Aj is the absorbance of the blank reagent.

| UHPLC-TOF/HRMS analysis of phenolic compounds
0.1 g freeze-dried sample was weighed. And 5 ml of 80% methanol aqueous solution was used to extract these samples with sonication for 1 h and filtered with a 0.2 μm membrane filter. An Ultimate 3000 RSLC UHPLC system (Thermo Fisher Scientific Inc.) was exposed to the phenolic compounds profiling of H. syriacus for the study.
The system consisted of an autosampler, a column oven, an ultrahigh pressure solvent delivery pump, and an automatic degasser.

| Statistical analysis
Triplicate analyses for each plant unit (leaves, petals, roots, and sprouts) were carried out. The obtained results were statistically processed using the program package SPSS 20. Statistical significance was considered at probability p < .01 based on Duncan's new multiple range test calculators. The mean values and standard deviations (SD) were calculated for all analyzed compounds. The data were as mean ± SE.

| Amino acid and fatty acid composition
Total amino acid contents of H. syriacus extracts from its different units (leaves, petals, sprouts, and roots) results from the test are presented in Table 1. Of a total of 20 amino acids, all but cysteine, 19 amino acids were isolated, including the ten essential amino acids.
The sum of amino acid content within each unit was highest in the sprouts (1274.6 mg/100 g), followed by the petals (737.2 mg/100 g), roots (581.5 mg/100 g), and leaves (277.1 mg/100 g). The total amino acid content of H. syriacus sprouts grown for 10 days was 1274.6 mg/100 g, lower than that of radish sprouts (Raphanus sativus L., 3020 mg/100 g) grown for 12 days, but produced a similar value to the 1313.1 mg/100 g from barley sprouts (Hordeum vulgare L.), a popular source for health food in Korea (Han et al., 2003;Son et al., 2016), grown for 10 days. To be used as food in the form of sprouts of Hibiscus syriacus, it should be superior in other excellent ingredients and amino acid content to the existing sprouts vegetables (radish, barley sprouts, and so on).
Among the 19 amino acids, seven amino acids, including alanine (171.6 ± 1.4 mg/100 g) and glutamine (101.2 ± 0.7 mg/100 g), were the highest content in the petals; these, also including glycine (295.3 ± 2.5 mg/100 g) and asparagine (268.1 ± 8.3 mg/100 g), yielded the highest in the sprouts (p < .01). Among the remaining amino acids, histidine, arginine, and tyrosine were the most abundant in the roots compared to the other units (p < .01). The essential amino acid content was high in the roots, at 317.6 mg/100 g, and sprouts, at 245.4 mg/100 g, with a significantly higher production of amino acids; arginine, at 274.4 mg/100 g, 134.6 mg/100 g, respectively. These results are significantly higher than radish sprouts (Raphanus sativus L, 120.7 mg/100 g) used as an ingredient in bibimbap, a traditional Korean food (Han et al., 2003). Arginine, which is converted into nitric oxide and citrulline in the body, improves circulation in the bloodstream. It acts as a vasodilator . Currently, many healthy functional foods containing arginine have been developed. It is worth examining whether the root, with the highest arginine content among the four parts, can be used for healthy food. Table 2 shows the total contents of the four fatty acids distributed by each unit as follows: sprouts (1070.0 mg/100 g), leaves (821.6 mg/100 g), roots (407.0 mg/100 g), petals (369.1 mg/100 g).
The sum of fatty acid content was as follows: linoleic acid (1311.2 mg/100 g), α-linolenic acid (663.1 mg/100 g), palmitic acid (592.6 mg/100 g), oleic acid (100.8 mg/100 g). Also, the ratio of unsaturated fatty acids (linoleic acid, α-linolenic acid, oleic acid) to saturated fatty acids (palmitic acid) was 77.8%-22.2% in total. Among the four types of fatty acids, except for α-linolenic acid, the sprouts had the highest content compared to the other units (p < .01). In Actinidia argute leaves, used as a spring vegetable in Korea, α-linolenic acid accounted for 66.8% of the total fatty acid content, linoleic acid was 13.8% as an unsaturated fatty acid, and palmitic acid accounted for 12.1% (Jin et al., 2015). In the flowers of Robinia pseudoacacia L., popular as an edible flower in Korea, palmitic acid represented TA B L E 1 Amino acid composition of each unit off H. syriacus (Unit: mg/100 g) (2) Total EAA: Total essential amino acid.
Bold letters mean the essential amino acid. ers every day for about 100 days, the seed yield is higher than other flowering trees. It will be easy to obtain the sprouts by cultivating these seeds within a short period.

| Free sugars and organic acids composition
The total free sugars contents of H. syriacus extracts from distinct parts of the plant (leaves, petals, sprouts, and roots) were tested with the data content presented in Table 3. The sum of the free sugar content within each unit was as follows: petals (9141.1 mg/100 g), leaves (2413.3 mg/100 g), sprouts (1429.3 mg/100 g), and roots (707.8 mg/100 g). The leaves contained five types of free sugars, while the petals had three, the sprouts two, and the roots one. The leaves contained glucose (847.9 mg/100 g) and raffinose (836.3 mg/100 g) presented similarly high values. Trisaccharide raffinose and tetrasaccharide stachyose were detected only in the leaves.
The petals contained high levels of fructose (4085.0 mg/100 g), glucose (3873.8 mg/100 g), and sucrose (1182.3 mg/100 g). The highest content of glucose and sucrose found in the petals was at (p < .01). The roots contained only sucrose (707.8 mg/100 g), and the sprouts contained both glucose (949.5 mg/100 g) and sucrose (479.8 mg/100 g). Although the free sugar content in H. syriacus petals was lower than that of Robinia pseudoacacia L. flowers (fructose 7500.2 mg/100 g, glucose 300.7 mg/100 g, sucrose 6100.7 mg/100 g), it was higher than that of Rhododendron mucronulatum Turcz. These are considered edible flowers during the spring season in Korea (Kwon et al., 1995). Table 4 shows the total organic acid content of each part of H. syriacus. The organic acid content of each part was as follows: leaves (524.9 mg/100 g), sprouts (508.0 mg/100 g), petals (460.9 mg/100 g), and roots (186.4 mg/100 g). The organic acids identified in H. syriacus were citric acid, fumaric acid, and malic acid.
The leaves and sprouts contained all three organic acids, while the petals contained only fumaric acid, and the roots contained citric acid and fumaric acid. Citric acid, a well-known natural ingredient extracted from sour fruits such as lemon and lime, is widely used as a preservative and is evenly contained in the leaves, roots, and sprouts of this plant, with the highest yield of 241.6 mg/100 g, found in the sprouts (Owen et al., 2002). The fumaric acid is found in each of the units, the leaves, petals, roots, and sprouts of H. syriacus, with the highest aggregate of 460.9 mg/100 g in petals. In comparison, the highest contents were those of fumaric acid (822.2 mg/100 g), citric acid (646.5 mg/100 g), and malic acid (211.5 mg/100 g). Fumaric acid, and its esters, possess the following: anti-inflammatory, hepatoprotective, analgesic, antitumor, and anti-intoxication activities, as well as strong antibacterial activity against Staphylococcus aureus,

| Total polyphenols content and antioxidant capacity
The result of the analysis of total polyphenol content of H. syriacus extracts within its different units (leaves, petals, roots, and sprouts) is in Figure 1. The phenolic compounds are secondary metabolites, widely distributed in plants systems, having various functions exhibiting a physiologically active functionality as an antioxidant. It is due to its strong binding properties to proteins and other macromolecules (AOAC, 1995). Among the total polyphenolic content of H. syriacus, leaves (1.18 ± 0.01%) and petals (1.15 ± 0.07%) showed the highest total value compared to the value of the sprouts (0.81 ± 0.04%) and roots (0.24 ± 0.01%).
The 70% EtOH extracts of each H. syriacus unit showed high antioxidant activity in ABTS free radical scavenging assays. At a low concentration of 25 μg/ml, the antioxidant activity within each unit was as follows: the highest value established at 56.4% for petals, 45.3% for leaves, 31.4% for sprouts, and 25.4% for roots, respectively ( Figure 2). Following these results, it is understood that the antioxidant activity was high in the petals with the highest polyphenol content. The calyx fruits of Hibiscus sabdariffa L., which is a plant of considerable medicinal and economical value worldwide as a genus of Hibiscus, showed less than 40% free radical scavenging activity in hot water extract and 30%-95% in EtOH extract at 200μg/mL which is a higher concentration (Yang et al., 2012).

| Identification of phenolic compounds by UHPLC-TOF/HRMS
A targeted analysis, based on UHPLC-TOF/HRMS (ultra-highperformance liquid chromatography-time-of-flight high-resolution mass spectrometry), was conducted to identify the phenolic compounds present in methanolic extracts of the units of H. syriacus since the phenolic composition of H. syriacus is still not reported in the literature. Table 5 shows that the phenolic profile was diverse among the leaves, petals, roots, and sprouts of H. syriacus.

| CON CLUS ION
This study highlights the abundance in the nutritional composition of each of the four units of H. syriacus commonly cultivated in Korea.
H. syriacus sprouts contain amino and fatty acids, which are of the highest aggregate in identifying their value as a functional food, and, since seeds are the means of propagation, sprouts are produced in abundance. H. syriacus petals contain a large number of free sugars, including fructose and sucrose, along with fumaric acid, which is why they are the most valuable as edible flowers or healthy functional foods having the highest number compared to the other parts.
Subsequently, 2 phenolic acids and 22 flavonoids, were also identified in the petals.
To conclude, the results generated in this study strongly suggest the possibility of excellent pharmacological activity and nutritional advantages, not only in the petals but across each division of the H. syriacus plant.

ACK N OWLED G EM ENTS
This study was supported by the National Institute of Forest Science, Korea (project number FG0403-2018-01), and by the National Research Foundation of Korea (NRF) funded by the Korean government.

CO N FLI C T O F I NTE R E S T
The author declares no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article.